Living cells convert nutrients to energy and various chemical byproducts through a series of oxidation and reduction reactions. By monitoring the byproducts excreted into, or taken up from, the extracellular medium, one can gain information about the underlying metabolic pathways and associated metabolic rates. This knowledge, in turn, can be useful in determining cell viability, studying the mechanisms underlying various diseases, and screening new drug candidates for effectiveness as well as unexpected or adverse effects. For example, the combined measurement of at least two of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and CO2 production rate (CPR) can discriminate between glucose and fatty acid oxidation, and thereby serve as a basis for testing drugs targeting obesity or diabetes. Similarly, extracellular flux rate measurements can be used in developing cancer therapies which exploit differences in the relative utilization of aerobic and anaerobic metabolic pathways between cancerous and non-cancerous cells.
Extracellular flux rates can be quantified with a variety of sensors, including, e.g., fluorescent sensors, ISFET sensors, and amperametric sensors such as the Clark electrode. For reliable detection, device sensitivity herein sets a lower bound for the analyte concentration, which translates into a minimal required cell density. Typically, cell densities sufficiently high for measurements are too high for maintaining cell viability and growth. This conflict can be resolved with apparatus that allow for low cell densities for cell maintenance, and temporarily increased densities during measurement. One such apparatus has been described in U.S. Pat. No. 7,276,351 (Teich et al., “Method and device for measuring multiple physiological properties of cells”), the disclosure of which is incorporated herein by reference. It utilizes a vessel holding cells in a medium, such as a microtiter well plate, and a plunger which can be immersed into a well to create a reduced volume for measurements.
Separating the byproducts of metabolic reactions from molecules of the same type, but different origin, that are present in the environment is another challenge encountered in measuring extracellular fluxes. Usually, analyte background flux is measured in blank (i.e., cell-free) sample, and subtracted from the fluxes measured in medium in the presence of cell cultures. For example, when using a well plate for cell-based assays, one of the wells may serve as the reference well. This approach produced satisfactory results, provided that the background rate is uniform across the plate and low compared with the total flux.